Toll-like receptors (TLRs) are a family of transmembrane proteins, which recognize structurally conserved molecules that are derived from pathogens, referred to as pathogen-associated molecular patterns. As such, TLRs function in the mammalian immune system as front-line sensors of pathogen-associated molecular patterns, detecting the presence of invading pathogens (Takeuchi and Akira 2010, Cell 140:805-820). The human genome contains 10 known TLRs; of these, TLR7 and TLR8 sense single stranded RNA ligands and their (oligo)nucleotide degradation products. The distribution of TLR7 and TLR8 is restricted to the endolysosomal compartment and the receptors are preferentially expressed in antigen presenting cells (APCs), a key cell type that modulates immune system activation.
TLR engagement in sentinel immune cells causes biosynthesis of selected cytokines (e.g., type I interferons), induction of co-stimulatory molecules, and increased antigen presentation capacity by APCs; important molecular mechanisms that activate innate and adaptive immune responses. Engagement of TLR7 in plasmacytoid dendritic cells leads to the induction of IFN-α/β, which plays essential functions in the control of adaptive immunity. TLR8 is expressed in myeloid dendritic cells, monocytes, and monocyte-derived dendritic cells and agonist engagement of TLR8 induces a prominent pro-inflammatory cytokine profile, characterized by increased production of tumor necrosis factor α (TNF-α), interleukin-12 (IL-12), and IL-18. Thus, virtually all major types of monocytic and dendritic cells can be activated by agonists of either TLR7 or TLR8 to become highly effective antigen-presenting cells. As most antigen presenting cell types express only one of these two receptors, agonists that can stimulate both receptors are potentially more effective adjuvants than agonists specific for only one of these TLR (Wille-Reece, et al. Proc. Nat'l Acad. Sci. 2005, 102:15190-15194). Additionally, TLR7 and TLR7/8 agonists can also be effective in stimulating anti-tumor immune responses in cancer, based on studies in animal models (Singh, et al. J Immunol 2014, 193:4722-4731). Accordingly, TLR agonists have been extensively investigated as stimulators of innate and adaptive immune responses, including for use as cancer therapeutic agents and vaccine adjuvants (Sabado et al. 2015, Ca Immunol Res 3:278-287; Vasilakos and Tomai 2013, Exp Rev Vaccines 12:809-819).
Although a number of small molecule structural classes are known to interact at the guanosine/uridine ligand binding site and possess varying levels of TLR7 and/or TLR8 agonist bioactivity, many such agonists are derivatives of the 1H-imidazo[4,5-c]quinoline privileged chemical template. One early example is 1-isobutyl-1H-imidazo[4,5-c]quinolin-4-amine (Imiquimod), a modest potency TLR7 agonist that was approved in 1997 as a topical formulation for actinic keratosis, superficial basal cell carcinoma, and genital warts. Subsequent medicinal chemistry efforts have produced several derivatives with significantly improved dual TLR7/8 agonist activity, most notably 1-(4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol (R848, Resiquimod), as well as 1-(4-aminomethylbenzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (IMDQ, FIG. 1) and 1-(3-(aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (meta-IMDQ, FIG. 1; see, e.g., Beesu, M. et al. 2015, J Med Chem 50:7833-7849; U.S. Pat. Nos. 8,728,486 and 9,441,005, incorporated herein by reference). However, rapid systemic diffusion of these small molecule compounds following local administration (e.g., subcutaneous [SC], intratumoral [IT], or intramuscular [IM]) of pharmacologically relevant doses, leads to systemic induction of pro-inflammatory cytokine responses, elevating the risk of adverse events in humans (e.g., fever, malaise, lymphopenia, etc.), see, e.g., Vasilakos and Tomai 2013, Exp Rev Vaccines 12:809-819; Smirnov, D. et al. 2011, Vaccine 29:5434-5442).
Therefore, there remains a need for immunotherapeutic agents that: 1) possess TLR7/8 agonist activity with potent bioactivity against both receptors and with potent immunostimulatory activities that activate larger subsets of APCs than exclusively TLR7 or TLR8 agonists alone; 2) preferentially target a stable prodrug form of the TLR7/8 agonist to the tumor microenvironment or locally retain a stable prodrug form of the TLR7/8 agonist at the tumor microenvironment following SC or intravenous administration, and subsequently release the active form within the tumor microenvironment; and 3) possess physiochemical properties that restrict subsequent distribution of the released, active form (i.e., unconjugated) of the TLR7/8 agonist from the tumor microenvironment.
The present invention provides for conjugates of TLR7/8 agonist compounds containing a cleavable linker and an agent for tissue-specific targeting or for local retention following administration, methods for preparation thereof, uses thereof for stimulating local immune responses and reducing unwanted systemic immune activation, and uses thereof for the treatment of cancer.